Methods for printing cells and generating arrays of barcoded cells

ABSTRACT

This disclosure relates to compositions and methods for analyzing single cells using cell printing and spatial analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/964,055, filed Jan. 21, 2020; which is incorporated herein byreference in its entirety.

BACKGROUND

Cells within a tissue of a subject have differences in cell morphologyand/or function due to varied analyte levels (e.g., gene and/or proteinexpression) within the different cells. The specific position of a cellwithin a tissue (e.g., the cell's position relative to neighboring cellsor the cell's position relative to the tissue microenvironment) canaffect, e.g., the cell's morphology, differentiation, fate, viability,proliferation, behavior, and signaling and cross-talk with other cellsin the tissue.

Spatial heterogeneity has been previously studied using techniques thatonly provide data for a small handful of analytes in the contact of anintact tissue or a portion of a tissue, or provide a lot of analyte datafor single cells, but fail to provide information regarding the positionof the single cell in a parent biological sample (e.g., tissue sample).

Genetic material, and related gene and protein expression, influencescellular fate and behavior. Analysis on a single cell-level will allowinsights into individual cell genotype and function. Others haveidentified methods of single cell isolation while maintaining viability.See, e.g., Zhang et al., PNAS, 2014 111 (8) 2948-2953; Marzo et al.,Nature Communications, 6, 8661 (2015); Laurell et al., Chem. Soc. Rev.,2007, 36, 492-506; and Ding et al., PNAS, 2012 109 (28) 11105-11109;each of which is incorporated by reference in its entirety. There,however, remains a need to develop extend these techniques using highthroughput techniques for genotypic and phenotypic single-cell analysis.

SUMMARY

Provided herein are methods for determining a location of an analyte ina cell, the methods include: (a) separating the cell from a plurality ofcells; (b) printing the cell onto a surface comprising an array, whereinthe array comprises a plurality of capture probes, wherein a captureprobe of the plurality of capture probes comprises: (i) a spatialbarcode and (ii) a capture domain; (c) hybridizing the analyte to thecapture domain; and (d) determining (i) all or a part of the sequence ofthe analyte bound to the capture domain, or a complement thereof, and(ii) all or a part of the sequence of the spatial barcode, or acomplement thereof, and using the determined sequence of (i) and (ii) toidentify the location of the analyte in the cell.

Also provided herein are methods for spatial profiling a biologicalanalyte in a cell that include: (a) separating the cell from a pluralityof cells; (b) printing the cell onto a surface; (c) contacting the cellwith a substrate comprising a plurality of capture probes, wherein acapture probe of the plurality comprises a spatial barcode and a capturedomain; (d) releasing the biological analyte from the cell, wherein thebiological analyte is bound by the capture probe at a distinct spatialposition of the substrate; (e) detecting the biological analyte bound bythe capture probe; and (f) correlating the biological analyte with thespatial barcode at the distinct spatial position of the substrate; thusprofiling the biological analyte as present in the cell at the distinctspatial position. In some embodiments, the step of separating the cellfrom a plurality of cells includes filtering a cell through a mold. Insome embodiments, the methods further include removing the mold prior tocontacting the cell with the substrate.

In some embodiments, the step of separating the cell from a plurality ofcells includes filtering a cell through a mold. Some embodiments of themethods described herein further include removing the mold afterprinting the cell onto a surface. In some embodiments, the plurality ofcells have at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% viability.In some embodiments, the cell is from a heterogeneous cell population.In some embodiments, the cell is from a formalin-fixed,paraffin-embedded (FFPE) sample, a frozen sample, or a fresh sample. Insome embodiments, the cell is from a tissue sample or a cell culturesample. In some embodiments, the surface includes glass, a modifiedglass, a functionalized glass, a hydrogel, a film, a membrane, aplastic, a nylon, a ceramic, a resin, Zeonor, silica, carbon, metals,inorganic glasses, optical fiber bundles, polymers, or combinationsthereof. In some embodiments, the mold includes a network of channelsthat allow the cell to migrate towards the surface. In some embodiments,the detecting step allows for resolution of 5 μm.

In some embodiments, the mold includes from about 2 to about 100individual channels in the network of channels. In some embodiments, themold comprises 2, 3, 4, or 5 individual channels in the network ofchannels. In some embodiments, the individual channels include trapspacing, wherein the trap spacings in individual channels proximal tothe surface are narrower in diameter than the trap spacings inindividual channels distal to the surface. In some embodiments, theanalyte is a protein. In some embodiments, the analyte includes apost-translational modification. In some embodiments, the analyte is aDNA or RNA. In some embodiments, the RNA is an mRNA. In someembodiments, the determining step includes amplifying all or part of theanalyte bound to the capture domain. In some embodiments, thedetermining step includes sequencing. Some embodiments of any of themethods described herein further include imaging the cell. In someembodiments, the imaging is used to determine the morphology of thecell. In some embodiments, the capture probe includes a unique molecularidentifier, a cleavage domain, and/or a functional domain.

In some embodiments, the determining step comprises amplifying all orpart of the ligated probe specifically bound to the capture domain. Insome embodiments, the amplifying is isothermal. In some embodiments, theamplifying is not isothermal. In some embodiments, an amplifying productcomprises (i) all or part of sequence of the ligated probe specificallybound to the capture domain, or a complement thereof, and (ii) all or apart of the sequence of the spatial barcode, or a complement thereof. Insome embodiments, the determining step comprises sequencing. In someembodiments, the sequencing is in situ sequencing. In some embodiments,in situ sequencing is performed via sequencing-by-synthesis (SBS),sequential fluorescence hybridization, sequencing by ligation, nucleicacid hybridization, or high-throughput digital sequencing techniques. Insome embodiments, the step of releasing the biological analyte comprisespermeabilizing the cell. In some embodiments, the methods furtherinclude fixing the cell prior to the permeabilizing the cell. In someembodiments, the methods further include staining the cell prior to thepermeabilizing the cell. In some embodiments, the cell is stained afterthe fixing the cell. In some embodiments, the cell is fixed andpermeabilized prior to releasing the biological analyte from thebiological sample. In some embodiments, permeabilizing the cellcomprises electrophoresis. In some embodiments, permeabilizing the cellcomprises administering a permeabilization reagent. In some embodiments,the methods further include imaging the cell. In some embodiments,imaging is performed prior to releasing the biological analyte from thecell. In some embodiments, imaging is performed after releasing thebiological analyte from the cell. In some embodiments, imaging is usedto determine the morphology of the cell. In some embodiments, thecapture probe comprises a unique molecular identifier. In someembodiments, the capture probe comprises a cleavage domain. In someembodiments, the capture probe comprises a functional domain. In someembodiments, the functional domain is a primer sequence. In someembodiments, the capture probe comprises a capture domain. In someembodiments, the capture domain comprises a poly-dT sequence. In someembodiments, the capture domain is configured to hybridize to a poly-Atail of an mRNA.

All publications, patents, patent applications, and informationavailable on the internet and mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication, patent, patent application, or item of information wasspecifically and individually indicated to be incorporated by reference.To the extent publications, patents, patent applications, and items ofinformation incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

Where values are described in terms of ranges, it should be understoodthat the description includes the disclosure of all possible sub-rangeswithin such ranges, as well as specific numerical values that fallwithin such ranges irrespective of whether a specific numerical value orspecific sub-range is expressly stated.

The term “each”, when used in reference to a collection of items, isintended to identify an individual item in the collection but does notnecessarily refer to every item in the collection, unless expresslystated otherwise, or unless the context of the usage clearly indicatesotherwise.

The singular form “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise. For example, the term “a cell”includes one or more cells, comprising mixtures thereof “A and/or B” isused herein to include all of the following alternatives: “A”, “B”, “Aor B”, and “A and B”.

Various embodiments of the features of this disclosure are describedherein. However, it should be understood that such embodiments areprovided merely by way of example, and numerous variations, changes, andsubstitutions can occur to those skilled in the art without departingfrom the scope of this disclosure. It should also be understood thatvarious alternatives to the specific embodiments described herein arealso within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the featuresand advantages of this disclosure. These embodiments are not intended tolimit the scope of the appended claims in any manner. Like referencesymbols in the drawings indicate like elements.

FIG. 1 is a schematic diagram showing an example of a barcoded captureprobe, as described herein.

FIG. 2 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to target analytes within the sample.

FIG. 3 is a schematic diagram of an exemplary multiplexedspatially-barcoded feature.

FIG. 4 is a schematic diagram of an exemplary analyte capture agent.

FIG. 5 is a schematic diagram depicting an exemplary interaction betweena feature-immobilized capture probe 524 and an analyte capture agent526.

FIGS. 6A, 6B, and 6C are schematics illustrating how streptavidin celltags can be utilized in an array-based system to produce aspatially-barcoded cells or cellular contents.

FIG. 7 shows an exemplary workflow for determining a location of ananalyte in a cell.

DETAILED DESCRIPTION I. Introduction

Disclosed herein are methods and compositions predicated on theidentification of methods of determining analyte expression in a singlecell. The methods disclosed herein include isolating single cellpopulations onto an array comprising a plurality of capture probes. Theability of printing single-cell arrays with high precision andefficiency, single-cell resolution, multiple cell types, and maintenanceof cell viability and function can be used to study cell function andeven population heterogeneity. Here, this type of single cell isolationis combined with spatial analysis techniques in order to determineabundance of one or more analytes in a single cell. The combinedmethods, which include imaging of a cell, allow for analysis of thecorrelation between phenotype and genotype in a cell; it addressesissues such as relating cell viability and sequencing data quality;doublet detection, and debris discard.

Spatial analysis methodologies and compositions described herein canprovide a vast amount of analyte and/or expression data for a variety ofanalytes within a biological sample at high spatial resolution, whileretaining native spatial context. Spatial analysis methods andcompositions can include, e.g., the use of a capture probe including aspatial barcode (e.g., a nucleic acid sequence that provides informationas to the location or position of an analyte within a cell or a tissuesample (e.g., mammalian cell or a mammalian tissue sample) and a capturedomain that is capable of binding to an analyte (e.g., a protein and/ora nucleic acid) produced by and/or present in a cell. Spatial analysismethods and compositions can also include the use of a capture probehaving a capture domain that captures an intermediate agent for indirectdetection of an analyte. For example, the intermediate agent can includea nucleic acid sequence (e.g., a barcode) associated with theintermediate agent. Detection of the intermediate agent is thereforeindicative of the analyte in the cell or tissue sample.

Non-limiting aspects of spatial analysis methodologies and compositionsare described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022,10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810,9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent ApplicationPublication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641,2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709,2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322,2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875,2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO2020/176788, Rodrigues et al., Science 363(6434):1463-1467, 2019; Lee etal., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gaoet al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol.36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits UserGuide (e.g., Rev C, dated June 2020), and/or the Visium Spatial TissueOptimization Reagent Kits User Guide (e.g., Rev C, dated July 2020),both of which are available at the 10× Genomics Support Documentationwebsite, and can be used herein in any combination. Further non-limitingaspects of spatial analysis methodologies and compositions are describedherein.

Some general terminology that may be used in this disclosure can befound in Section (I)(b) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Typically, a “barcode” is a label, oridentifier, that conveys or is capable of conveying information (e.g.,information about an analyte in a sample, a bead, and/or a captureprobe). A barcode can be part of an analyte, or independent of ananalyte. A barcode can be attached to an analyte. A particular barcodecan be unique relative to other barcodes. For the purpose of thisdisclosure, an “analyte” can include any biological substance,structure, moiety, or component to be analyzed. The term “target” cansimilarly refer to an analyte of interest.

Analytes can be broadly classified into one of two groups: nucleic acidanalytes, and non-nucleic acid analytes. Examples of non-nucleic acidanalytes include, but are not limited to, lipids, carbohydrates,peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins,phosphoproteins, specific phosphorylated or acetylated variants ofproteins, amidation variants of proteins, hydroxylation variants ofproteins, methylation variants of proteins, ubiquitylation variants ofproteins, sulfation variants of proteins, viral proteins (e.g., viralcapsid, viral envelope, viral coat, viral accessory, viralglycoproteins, viral spike, etc.), extracellular and intracellularproteins, antibodies, and antigen binding fragments. In someembodiments, the analyte(s) can be localized to subcellular location(s),including, for example, organelles, e.g., mitochondria, Golgi apparatus,endoplasmic reticulum, chloroplasts, endocytic vesicles, exocyticvesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) canbe peptides or proteins, including without limitation antibodies andenzymes. Additional examples of analytes can be found in Section (I)(c)of WO 2020/176788 and/or U.S. Patent Application Publication No.2020/0277663. In some embodiments, an analyte can be detectedindirectly, such as through detection of an intermediate agent, forexample, a connected probe (e.g., a ligation product) or an analytecapture agent (e.g., an oligonucleotide-conjugated antibody), such asthose described herein.

A “biological sample” is typically obtained from the subject foranalysis using any of a variety of techniques including, but not limitedto, biopsy, surgery, and laser capture microscopy (LCM), and generallyincludes cells and/or other biological material from the subject. Insome embodiments, a biological sample can be a tissue section. In someembodiments, a biological sample can be a fixed and/or stainedbiological sample (e.g., a fixed and/or stained tissue section).Non-limiting examples of stains include histological stains (e.g.,hematoxylin and/or eosin) and immunological stains (e.g., fluorescentstains). In some embodiments, a biological sample (e.g., a fixed and/orstained biological sample) can be imaged. Biological samples are alsodescribed in Section (I)(d) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, a biological sample is permeabilized with one ormore permeabilization reagents. For example, permeabilization of abiological sample can facilitate analyte capture. Exemplarypermeabilization agents and conditions are described in Section(I)(d)(ii)(13) or the Exemplary Embodiments Section of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

Array-based spatial analysis methods involve the transfer of one or moreanalytes from a biological sample to an array of features on asubstrate, where each feature is associated with a unique spatiallocation on the array. Subsequent analysis of the transferred analytesincludes determining the identity of the analytes and the spatiallocation of the analytes within the biological sample. The spatiallocation of an analyte within the biological sample is determined basedon the feature to which the analyte is bound (e.g., directly orindirectly) on the array, and the feature's relative spatial locationwithin the array.

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte (e.g., an analyte ofinterest) in a biological sample. In some embodiments, the capture probeis a nucleic acid or a polypeptide. In some embodiments, the captureprobe includes a barcode (e.g., a spatial barcode and/or a uniquemolecular identifier (UMI)) and a capture domain). In some embodiments,a capture probe can include a cleavage domain and/or a functional domain(e.g., a primer-binding site, such as for next-generation sequencing(NGS)).

FIG. 1 is a schematic diagram showing an exemplary capture probe, asdescribed herein. As shown, the capture probe 102 is optionally coupledto a feature 101 by a cleavage domain 103, such as a disulfide linker.The capture probe can include a functional sequence 104 that is usefulfor subsequent processing. The functional sequence 104 can include allor a part of sequencer specific flow cell attachment sequence (e.g., aP5 or P7 sequence), all or a part of a sequencing primer sequence,(e.g., a R1 primer binding site, a R2 primer binding site), orcombinations thereof. The capture probe can also include a spatialbarcode 105. The capture probe can also include a unique molecularidentifier (UMI) sequence 106. While FIG. 1 shows the spatial barcode105 as being located upstream (5′) of UMI sequence 106, it is to beunderstood that capture probes wherein UMI sequence 106 is locatedupstream (5′) of the spatial barcode 105 is also suitable for use in anyof the methods described herein. The capture probe can also include acapture domain 107 to facilitate capture of a target analyte. Thecapture domain can have a sequence complementary to a sequence of anucleic acid analyte. The capture domain can have a sequencecomplementary to a connected probe described herein. The capture domaincan have a sequence complementary to a capture handle sequence presentin an analyte capture agent. The capture domain can have a sequencecomplementary to a splint oligonucleotide. Such splint oligonucleotide,in addition to having a sequence complementary to a capture domain of acapture probe, can have a sequence of a nucleic acid analyte, a sequencecomplementary to a portion of a connected probe described herein, and/ora capture handle sequence described herein.

The functional sequences can generally be selected for compatibilitywith any of a variety of different sequencing systems, e.g., Ion TorrentProton or PGM, Illumina sequencing instruments, PacBio, Oxford Nanopore,etc., and the requirements thereof. In some embodiments, functionalsequences can be selected for compatibility with non-commercializedsequencing systems. Examples of such sequencing systems and techniques,for which suitable functional sequences can be used, include (but arenot limited to) Ion Torrent Proton or PGM sequencing, Illuminasequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing.Further, in some embodiments, functional sequences can be selected forcompatibility with other sequencing systems, includingnon-commercialized sequencing systems.

In some embodiments, the spatial barcode 105 and functional sequences104 are common to all of the probes attached to a given feature. In someembodiments, the UMI sequence 106 of a capture probe attached to a givenfeature is different from the UMI sequence of a different capture probeattached to the given feature.

FIG. 2 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to analytes within the sample. The capture probe 201 contains acleavage domain 202, a cell penetrating peptide 203, a reporter molecule204, and a disulfide bond (—S—S—). 205 represents all other parts of acapture probe, for example a spatial barcode and a capture domain.

FIG. 3 is a schematic diagram of an exemplary multiplexedspatially-barcoded feature. In FIG. 3 , the feature 301 can be coupledto spatially-barcoded capture probes, wherein the spatially-barcodedprobes of a particular feature can possess the same spatial barcode, buthave different capture domains designed to associate the spatial barcodeof the feature with more than one target analyte. For example, a featuremay be coupled to four different types of spatially-barcoded captureprobes, each type of spatially-barcoded capture probe possessing thespatial barcode 302. One type of capture probe associated with thefeature includes the spatial barcode 302 in combination with a poly(T)capture domain 303, designed to capture mRNA target analytes. A secondtype of capture probe associated with the feature includes the spatialbarcode 302 in combination with a random N-mer capture domain 304 forgDNA analysis. A third type of capture probe associated with the featureincludes the spatial barcode 302 in combination with a capture domaincomplementary to a capture handle sequence of an analyte capture agentof interest 305. A fourth type of capture probe associated with thefeature includes the spatial barcode 302 in combination with a capturedomain that can specifically bind a nucleic acid molecule 306 that canfunction in a CRISPR assay (e.g., CRISPR/Cas9). While only fourdifferent capture probe-barcoded constructs are shown in FIG. 3 ,capture-probe barcoded constructs can be tailored for analyses of anygiven analyte associated with a nucleic acid and capable of binding withsuch a construct. For example, the schemes shown in FIG. 3 can also beused for concurrent analysis of other analytes disclosed herein,including, but not limited to: (a) mRNA, a lineage tracing construct,cell surface or intracellular proteins and metabolites, and gDNA; (b)mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq)cell surface or intracellular proteins and metabolites, and aperturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein); (c)mRNA, cell surface or intracellular proteins and/or metabolites, abarcoded labelling agent (e.g., the MHC multimers described herein), anda V(D)J sequence of an immune cell receptor (e.g., T-cell receptor). Insome embodiments, a perturbation agent can be a small molecule, anantibody, a drug, an aptamer, a miRNA, a physical environmental (e.g.,temperature change), or any other known perturbation agents. See, e.g.,Section (II)(b) (e.g., subsections (i)-(vi)) of WO 2020/176788 and/orU.S. Patent Application Publication No. 2020/0277663. Generation ofcapture probes can be achieved by any appropriate method, includingthose described in Section (II)(d)(ii) of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663.

In some embodiments, more than one analyte type (e.g., nucleic acids andproteins) from a biological sample can be detected (e.g., simultaneouslyor sequentially) using any appropriate multiplexing technique, such asthose described in Section (IV) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, detection of one or more analytes (e.g., proteinanalytes) can be performed using one or more analyte capture agents. Asused herein, an “analyte capture agent” refers to an agent thatinteracts with an analyte (e.g., an analyte in a biological sample) andwith a capture probe (e.g., a capture probe attached to a substrate or afeature) to identify the analyte. In some embodiments, the analytecapture agent includes: (i) an analyte binding moiety (e.g., that bindsto an analyte), for example, an antibody or antigen-binding fragmentthereof; (ii) analyte binding moiety barcode; and (iii) a capture handlesequence. As used herein, the term “analyte binding moiety barcode”refers to a barcode that is associated with or otherwise identifies theanalyte binding moiety. As used herein, the term “analyte capturesequence” or “capture handle sequence” refers to a region or moietyconfigured to hybridize to, bind to, couple to, or otherwise interactwith a capture domain of a capture probe. In some embodiments, a capturehandle sequence is complementary to a capture domain of a capture probe.In some cases, an analyte binding moiety barcode (or portion thereof)may be able to be removed (e.g., cleaved) from the analyte captureagent.

FIG. 4 is a schematic diagram of an exemplary analyte capture agent 402comprised of an analyte-binding moiety 404 and an analyte-binding moietybarcode domain 408. The exemplary analyte-binding moiety 404 is amolecule capable of binding to an analyte 406 and the analyte captureagent is capable of interacting with a spatially-barcoded capture probe.The analyte-binding moiety can bind to the analyte 406 with highaffinity and/or with high specificity. The analyte capture agent caninclude an analyte-binding moiety barcode domain 408, a nucleotidesequence (e.g., an oligonucleotide), which can hybridize to at least aportion or an entirety of a capture domain of a capture probe. Theanalyte-binding moiety barcode domain 408 can comprise an analytebinding moiety barcode and a capture handle sequence described herein.The analyte-binding moiety 404 can include a polypeptide and/or anaptamer. The analyte-binding moiety 404 can include an antibody orantibody fragment (e.g., an antigen-binding fragment).

FIG. 5 is a schematic diagram depicting an exemplary interaction betweena feature-immobilized capture probe 524 and an analyte capture agent526. The feature-immobilized capture probe 524 can include a spatialbarcode 508 as well as functional sequences 506 and UMI 510, asdescribed elsewhere herein. The capture probe can also include a capturedomain 512 that is capable of binding to an analyte capture agent 526.The analyte capture agent 526 can include a functional sequence 518,analyte binding moiety barcode 516, and a capture handle sequence 514that is capable of binding to the capture domain 512 of the captureprobe 524. The analyte capture agent can also include a linker 520 thatallows the capture agent barcode domain 516 to couple to the analytebinding moiety 522.

FIGS. 6A, 6B, and 6C are schematics illustrating how streptavidin celltags can be utilized in an array-based system to produce aspatially-barcoded cell or cellular contents. For example, as shown inFIG. 6A, peptide-bound major histocompatibility complex (MHC) can beindividually associated with biotin (β2m) and bound to a streptavidinmoiety such that the streptavidin moiety comprises multiple pMHCmoieties. Each of these moieties can bind to a TCR such that thestreptavidin binds to a target T-cell via multiple MHC/TCR bindinginteractions. Multiple interactions synergize and can substantiallyimprove binding affinity. Such improved affinity can improve labellingof T-cells and also reduce the likelihood that labels will dissociatefrom T-cell surfaces. As shown in FIG. 6B, a capture agent barcodedomain 601 can be modified with streptavidin 602 and contacted withmultiple molecules of biotinylated MHC 603 such that the biotinylatedMHC 603 molecules are coupled with the streptavidin conjugated captureagent barcode domain 601. The result is a barcoded MHC multimer complex605. As shown in FIG. 6B, the capture agent barcode domain sequence 601can identify the MHC as its associated label and also includes optionalfunctional sequences such as sequences for hybridization with otheroligonucleotides. As shown in FIG. 6C, one example oligonucleotide iscapture probe 606 that comprises a complementary sequence (e.g., rGrGrGcorresponding to C C C), a barcode sequence and other functionalsequences, such as, for example, a UMI, an adapter sequence (e.g.,comprising a sequencing primer sequence (e.g., R1 or a partial R1(“pR1”), R2), a flow cell attachment sequence (e.g., P5 or P7 or partialsequences thereof)), etc. In some cases, capture probe 606 may at firstbe associated with a feature (e.g., a gel bead) and released from thefeature. In other embodiments, capture probe 606 can hybridize with acapture agent barcode domain 601 of the MHC-oligonucleotide complex 605.The hybridized oligonucleotides (Spacer C C C and Spacer rGrGrG) canthen be extended in primer extension reactions such that constructscomprising sequences that correspond to each of the two spatial barcodesequences (the spatial barcode associated with the capture probe, andthe barcode associated with the MHC-oligonucleotide complex) aregenerated. In some cases, one or both of these corresponding sequencesmay be a complement of the original sequence in capture probe 606 orcapture agent barcode domain 601. In other embodiments, the captureprobe and the capture agent barcode domain are ligated together. Theresulting constructs can be optionally further processed (e.g., to addany additional sequences and/or for clean-up) and subjected tosequencing. As described elsewhere herein, a sequence derived from thecapture probe 606 spatial barcode sequence may be used to identify afeature and the sequence derived from spatial barcode sequence on thecapture agent barcode domain 601 may be used to identify the particularpeptide WIC complex 604 bound on the surface of the cell (e.g., whenusing WIC-peptide libraries for screening immune cells or immune cellpopulations).

Additional description of analyte capture agents can be found in Section(II)(b)(ix) of WO 2020/176788 and/or Section (II)(b)(viii) U.S. PatentApplication Publication No. 2020/0277663.

There are at least two methods to associate a spatial barcode with oneor more neighboring cells, such that the spatial barcode identifies theone or more cells, and/or contents of the one or more cells, asassociated with a particular spatial location. One method is to promoteanalytes or analyte proxies (e.g., intermediate agents) out of a celland towards a spatially-barcoded array (e.g., includingspatially-barcoded capture probes). Another method is to cleavespatially-barcoded capture probes from an array and promote thespatially-barcoded capture probes towards and/or into or onto thebiological sample.

In some cases, capture probes may be configured to prime, replicate, andconsequently yield optionally barcoded extension products from atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent (e.g., a connected probe (e.g., a ligation product)or an analyte capture agent), or a portion thereof), or derivativesthereof (see, e.g., Section (II)(b)(vii) of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663 regarding extendedcapture probes). In some cases, capture probes may be configured to forma connected probe (e.g., a ligation product) with a template (e.g., aDNA or RNA template, such as an analyte or an intermediate agent, orportion thereof), thereby creating ligations products that serve asproxies for a template.

As used herein, an “extended capture probe” refers to a capture probehaving additional nucleotides added to the terminus (e.g., 3′ or 5′ end)of the capture probe thereby extending the overall length of the captureprobe. For example, an “extended 3′ end” indicates additionalnucleotides were added to the most 3′ nucleotide of the capture probe toextend the length of the capture probe, for example, by polymerizationreactions used to extend nucleic acid molecules including templatedpolymerization catalyzed by a polymerase (e.g., a DNA polymerase or areverse transcriptase). In some embodiments, extending the capture probeincludes adding to a 3′ end of a capture probe a nucleic acid sequencethat is complementary to a nucleic acid sequence of an analyte orintermediate agent specifically bound to the capture domain of thecapture probe. In some embodiments, the capture probe is extended usingreverse transcription. In some embodiments, the capture probe isextended using one or more DNA polymerases. The extended capture probesinclude the sequence of the capture probe and the sequence of thespatial barcode of the capture probe.

In some embodiments, extended capture probes are amplified (e.g., inbulk solution or on the array) to yield quantities that are sufficientfor downstream analysis, e.g., via DNA sequencing. In some embodiments,extended capture probes (e.g., DNA molecules) act as templates for anamplification reaction (e.g., a polymerase chain reaction).

Additional variants of spatial analysis methods, including in someembodiments, an imaging step, are described in Section (II)(a) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Analysis of captured analytes (and/or intermediate agents or portionsthereof), for example, including sample removal, extension of captureprobes, sequencing (e.g., of a cleaved extended capture probe and/or acDNA molecule complementary to an extended capture probe), sequencing onthe array (e.g., using, for example, in situ hybridization or in situligation approaches), temporal analysis, and/or proximity capture, isdescribed in Section (II)(g) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663. Some quality control measuresare described in Section (II)(h) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Spatial information can provide information of biological and/or medicalimportance. For example, the methods and compositions described hereincan allow for: identification of one or more biomarkers (e.g.,diagnostic, prognostic, and/or for determination of efficacy of atreatment) of a disease or disorder; identification of a candidate drugtarget for treatment of a disease or disorder; identification (e.g.,diagnosis) of a subject as having a disease or disorder; identificationof stage and/or prognosis of a disease or disorder in a subject;identification of a subject as having an increased likelihood ofdeveloping a disease or disorder; monitoring of progression of a diseaseor disorder in a subject; determination of efficacy of a treatment of adisease or disorder in a subject; identification of a patientsubpopulation for which a treatment is effective for a disease ordisorder; modification of a treatment of a subject with a disease ordisorder; selection of a subject for participation in a clinical trial;and/or selection of a treatment for a subject with a disease ordisorder.

Spatial information can provide information of biological importance.For example, the methods and compositions described herein can allowfor: identification of transcriptome and/or proteome expression profiles(e.g., in healthy and/or diseased tissue); identification of multipleanalyte types in close proximity (e.g., nearest neighbor analysis);determination of up- and/or down-regulated genes and/or proteins indiseased tissue; characterization of tumor microenvironments;characterization of tumor immune responses; characterization of cellstypes and their co-localization in tissue; and identification of geneticvariants within tissues (e.g., based on gene and/or protein expressionprofiles associated with specific disease or disorder biomarkers).

Typically, for spatial array-based methods, a substrate functions as asupport for direct or indirect attachment of capture probes to featuresof the array. A “feature” is an entity that acts as a support orrepository for various molecular entities used in spatial analysis. Insome embodiments, some or all of the features in an array arefunctionalized for analyte capture. Exemplary substrates are describedin Section (II)(c) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Exemplary features and geometricattributes of an array can be found in Sections (II)(d)(i),(II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Generally, analytes and/or intermediate agents (or portions thereof) canbe captured when contacting a biological sample with a substrateincluding capture probes (e.g., a substrate with capture probesembedded, spotted, printed, fabricated on the substrate, or a substratewith features (e.g., beads, wells) comprising capture probes). As usedherein, “contact,” “contacted,” and/or “contacting,” a biological samplewith a substrate refers to any contact (e.g., direct or indirect) suchthat capture probes can interact (e.g., bind covalently ornon-covalently (e.g., hybridize)) with analytes from the biologicalsample. Capture can be achieved actively (e.g., using electrophoresis)or passively (e.g., using diffusion). Analyte capture is furtherdescribed in Section (II)(e) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by attaching and/orintroducing a molecule (e.g., a peptide, a lipid, or a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) to a biologicalsample (e.g., to a cell in a biological sample). In some embodiments, aplurality of molecules (e.g., a plurality of nucleic acid molecules)having a plurality of barcodes (e.g., a plurality of spatial barcodes)are introduced to a biological sample (e.g., to a plurality of cells ina biological sample) for use in spatial analysis. In some embodiments,after attaching and/or introducing a molecule having a barcode to abiological sample, the biological sample can be physically separated(e.g., dissociated) into single cells or cell groups for analysis. Somesuch methods of spatial analysis are described in Section (III) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by detecting multipleoligonucleotides that hybridize to an analyte. In some instances, forexample, spatial analysis can be performed using RNA-templated ligation(RTL). Methods of RTL have been described previously. See, e.g., Credleet al., Nucleic Acids Res. 2017 Aug. 21; 45(14):e128. Typically, RTLincludes hybridization of two oligonucleotides to adjacent sequences onan analyte (e.g., an RNA molecule, such as an mRNA molecule). In someinstances, the oligonucleotides are DNA molecules. In some instances,one of the oligonucleotides includes at least two ribonucleic acid basesat the 3′ end and/or the other oligonucleotide includes a phosphorylatednucleotide at the 5′ end. In some instances, one of the twooligonucleotides includes a capture domain (e.g., a poly(A) sequence, anon-homopolymeric sequence). After hybridization to the analyte, aligase (e.g., SplintR ligase) ligates the two oligonucleotides together,creating a connected probe (e.g., a ligation product). In someinstances, the two oligonucleotides hybridize to sequences that are notadjacent to one another. For example, hybridization of the twooligonucleotides creates a gap between the hybridized oligonucleotides.In some instances, a polymerase (e.g., a DNA polymerase) can extend oneof the oligonucleotides prior to ligation. After ligation, the connectedprobe (e.g., a ligation product) is released from the analyte. In someinstances, the connected probe (e.g., a ligation product) is releasedusing an endonuclease (e.g., RNAse H). The released connected probe(e.g., a ligation product) can then be captured by capture probes (e.g.,instead of direct capture of an analyte) on an array, optionallyamplified, and sequenced, thus determining the location and optionallythe abundance of the analyte in the biological sample.

During analysis of spatial information, sequence information for aspatial barcode associated with an analyte is obtained, and the sequenceinformation can be used to provide information about the spatialdistribution of the analyte in the biological sample. Various methodscan be used to obtain the spatial information. In some embodiments,specific capture probes and the analytes they capture are associatedwith specific locations in an array of features on a substrate. Forexample, specific spatial barcodes can be associated with specific arraylocations prior to array fabrication, and the sequences of the spatialbarcodes can be stored (e.g., in a database) along with specific arraylocation information, so that each spatial barcode uniquely maps to aparticular array location.

Alternatively, specific spatial barcodes can be deposited atpredetermined locations in an array of features during fabrication suchthat at each location, only one type of spatial barcode is present sothat spatial barcodes are uniquely associated with a single feature ofthe array. Where necessary, the arrays can be decoded using any of themethods described herein so that spatial barcodes are uniquelyassociated with array feature locations, and this mapping can be storedas described above.

When sequence information is obtained for capture probes and/or analytesduring analysis of spatial information, the locations of the captureprobes and/or analytes can be determined by referring to the storedinformation that uniquely associates each spatial barcode with an arrayfeature location. In this manner, specific capture probes and capturedanalytes are associated with specific locations in the array offeatures. Each array feature location represents a position relative toa coordinate reference point (e.g., an array location, a fiducialmarker) for the array. Accordingly, each feature location has an“address” or location in the coordinate space of the array.

Some exemplary spatial analysis workflows are described in the ExemplaryEmbodiments section of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. See, for example, the Exemplary embodimentstarting with “In some non-limiting examples of the workflows describedherein, the sample can be immersed . . . ” of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663. See also, e.g., theVisium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C,dated June 2020), and/or the Visium Spatial Tissue Optimization ReagentKits User Guide (e.g., Rev C, dated July 2020).

In some embodiments, spatial analysis can be performed using dedicatedhardware and/or software, such as any of the systems described inSections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663, or any of one or more of thedevices or methods described in Sections Control Slide for Imaging,Methods of Using Control Slides and Substrates for, Systems of UsingControl Slides and Substrates for Imaging, and/or Sample and ArrayAlignment Devices and Methods, Informational labels of WO 2020/123320.

Suitable systems for performing spatial analysis can include componentssuch as a chamber (e.g., a flow cell or sealable, fluid-tight chamber)for containing a biological sample. The biological sample can be mountedfor example, in a biological sample holder. One or more fluid chamberscan be connected to the chamber and/or the sample holder via fluidconduits, and fluids can be delivered into the chamber and/or sampleholder via fluidic pumps, vacuum sources, or other devices coupled tothe fluid conduits that create a pressure gradient to drive fluid flow.One or more valves can also be connected to fluid conduits to regulatethe flow of reagents from reservoirs to the chamber and/or sampleholder.

The systems can optionally include a control unit that includes one ormore electronic processors, an input interface, an output interface(such as a display), and a storage unit (e.g., a solid state storagemedium such as, but not limited to, a magnetic, optical, or other solidstate, persistent, writeable and/or re-writeable storage medium). Thecontrol unit can optionally be connected to one or more remote devicesvia a network. The control unit (and components thereof) can generallyperform any of the steps and functions described herein. Where thesystem is connected to a remote device, the remote device (or devices)can perform any of the steps or features described herein. The systemscan optionally include one or more detectors (e.g., CCD, CMOS) used tocapture images. The systems can also optionally include one or morelight sources (e.g., LED-based, diode-based, lasers) for illuminating asample, a substrate with features, analytes from a biological samplecaptured on a substrate, and various control and calibration media.

The systems can optionally include software instructions encoded and/orimplemented in one or more of tangible storage media and hardwarecomponents such as application specific integrated circuits. Thesoftware instructions, when executed by a control unit (and inparticular, an electronic processor) or an integrated circuit, can causethe control unit, integrated circuit, or other component executing thesoftware instructions to perform any of the method steps or functionsdescribed herein.

In some cases, the systems described herein can detect (e.g., registeran image) the biological sample on the array. Exemplary methods todetect the biological sample on an array are described in PCTApplication No. 2020/061064 and/or U.S. patent application Ser. No.16/951,854.

Prior to transferring analytes from the biological sample to the arrayof features on the substrate, the biological sample can be aligned withthe array. Alignment of a biological sample and an array of featuresincluding capture probes can facilitate spatial analysis, which can beused to detect differences in analyte presence and/or level withindifferent positions in the biological sample, for example, to generate athree-dimensional map of the analyte presence and/or level. Exemplarymethods to generate a two- and/or three-dimensional map of the analytepresence and/or level are described in PCT Application No. 2020/053655and spatial analysis methods are generally described in WO 2020/061108and/or U.S. patent application Ser. No. 16/951,864.

In some cases, a map of analyte presence and/or level can be aligned toan image of a biological sample using one or more fiducial markers,e.g., objects placed in the field of view of an imaging system whichappear in the image produced, as described in the Substrate AttributesSection, Control Slide for Imaging Section of WO 2020/123320, PCTApplication No. 2020/061066, and/or U.S. patent application Ser. No.16/951,843. Fiducial markers can be used as a point of reference ormeasurement scale for alignment (e.g., to align a sample and an array,to align two substrates, to determine a location of a sample or array ona substrate relative to a fiducial marker) and/or for quantitativemeasurements of sizes and/or distances.

II. Cell Printing

(a) Biological Samples and Cells

A “biological sample” is obtained from the subject for analysis usingany of a variety of techniques including, but not limited to, biopsy,surgery, and laser capture microscopy (LCM), and generally includescells and/or other biological material from the subject. In addition tothe subjects described above, a biological sample can be obtained fromnon-mammalian organisms (e.g., a plant, an insect, an arachnid, anematode (e.g., Caenorhabditis elegans), a fungi, an amphibian, or afish (e.g., zebrafish)). A biological sample can be obtained from aprokaryote such as a bacterium, e.g., Escherichia coli, Staphylococci orMycoplasma pneumoniae; an archaea; a virus such as Hepatitis C virus orhuman immunodeficiency virus; or a viroid. A biological sample can beobtained from a eukaryote, such as a patient derived organoid (PDO) orpatient derived xenograft (PDX). The biological sample can includeorganoids, a miniaturized and simplified version of an organ produced invitro in three dimensions that shows realistic micro-anatomy. Organoidscan be generated from one or more cells from a tissue, embryonic stemcells, and/or induced pluripotent stem cells, which can self-organize inthree-dimensional culture owing to their self-renewal anddifferentiation capacities. In some embodiments, an organoid is acerebral organoid, an intestinal organoid, a stomach organoid, a lingualorganoid, a thyroid organoid, a thymic organoid, a testicular organoid,a hepatic organoid, a pancreatic organoid, an epithelial organoid, alung organoid, a kidney organoid, a gastruloid, a cardiac organoid, or aretinal organoid. Subjects from which biological samples can be obtainedcan be healthy or asymptomatic individuals, individuals that have or aresuspected of having a disease (e.g., cancer) or a pre-disposition to adisease, and/or individuals that are in need of therapy or suspected ofneeding therapy.

Biological samples can be derived from a homogeneous culture orpopulation of the subjects or organisms mentioned herein oralternatively from a collection of several different organisms, forexample, in a community or ecosystem.

Biological samples can include one or more diseased cells. A diseasedcell can have altered metabolic properties, gene expression, proteinexpression, and/or morphologic features. Examples of diseases includeinflammatory disorders, metabolic disorders, nervous system disorders,and cancer. Cancer cells can be derived from solid tumors, hematologicalmalignancies, cell lines, or obtained as circulating tumor cells.

Biological samples can also include fetal cells. For example, aprocedure such as amniocentesis can be performed to obtain a fetal cellsample from maternal circulation. Sequencing of fetal cells can be usedto identify any of a number of genetic disorders, including, e.g.,aneuploidy such as Down's syndrome, Edwards syndrome, and Patausyndrome. Further, cell surface features of fetal cells can be used toidentify any of a number of disorders or diseases.

Biological samples can also include immune cells. Sequence analysis ofthe immune repertoire of such cells, including genomic, proteomic, andcell surface features, can provide a wealth of information to facilitatean understanding of the status and function of the immune system. By wayof example, determining the status (e.g., negative or positive) ofminimal residue disease (MRD) in a multiple myeloma (MM) patientfollowing autologous stem cell transplantation is considered a predictorof MRD in the MM patient (see, e.g., U.S. Patent Application PublicationNo. 2018/0156784, the entire contents of which are incorporated hereinby reference).

Examples of immune cells in a biological sample include, but are notlimited to, B cells, T cells (e.g., cytotoxic T cells, natural killer Tcells, regulatory T cells, and T helper cells), natural killer cells,cytokine induced killer (CIK) cells, myeloid cells, such as granulocytes(basophil granulocytes, eosinophil granulocytes, neutrophilgranulocytes/hypersegmented neutrophils), monocytes/macrophages, mastcells, thrombocytes/megakaryocytes, and dendritic cells.

The biological sample can include any number of macromolecules, forexample, cellular macromolecules and organelles (e.g., mitochondria andnuclei). The biological sample can be a nucleic acid sample and/orprotein sample. The biological sample can be a carbohydrate sample or alipid sample. The biological sample can be obtained as a tissue sample,such as a tissue section, biopsy, a core biopsy, needle aspirate, orfine needle aspirate. The sample can be a fluid sample, such as a bloodsample, urine sample, or saliva sample. The sample can be a skin sample,a colon sample, a cheek swab, a histology sample, a histopathologysample, a plasma or serum sample, a tumor sample, living cells, culturedcells, a clinical sample such as, for example, whole blood orblood-derived products, blood cells, or cultured tissues or cells,including cell suspensions.

Cell-free biological samples can include extracellular polynucleotides.Extracellular polynucleotides can be isolated from a bodily sample,e.g., blood, plasma, serum, urine, saliva, mucosal excretions, sputum,stool, and tears.

As discussed above, a biological sample can include a single analyte ofinterest, or more than one analyte of interest. Methods for performingmultiplexed assays to analyze two or more different analytes in a singlebiological sample is discussed in a subsequent section of thisdisclosure.

The plurality of cells provided herein can be a homogenous orheterogeneous cell population. In some embodiments, the cells can beobtained from a tissue sample, a cell culture sample, or a body fluidsample. In some embodiments, the samples can be a formalin-fixed,paraffin-embedded sample, a frozen sample, or a fresh sample.

In some embodiments, cells separated and printed on a surface using themethods described herein can have a viability of at least 80% (e.g., atleast 81%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%). In some instances, thecells have 100% viability. In some embodiments, the cells can be viablefor at least 30 minutes (e.g. at least 45, 60, 75, 90, or 120 minutes)after being printed on the surface. In some instances, at least 50% ofthe cells (e.g, at least 55%, 60%, 75%, 80%, 85%, 90%, 95% of the cells)are alive two hours after being printed on the surface. Cell viabilityas described herein is a measure of the proportion of live healthy cellswithin a population of cells, e.g. using cell viability essays. Cellviability essays are well known in the art, such as but not limited to:TUNEL assay, Trypan Blue staining, Propidium iodide staining, and cellmembrane leakage assays.

In some embodiments, the cells provided herein can have a diameter ofabout 5 μm to about 25 μm (e.g., about 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 22, or 24 μm). The methods provided herein mayalso include staining the cell prior to separating the cell from theplurality of cells. The cells can be stained using techniques describedherein, such as fluorescent labeling.

In some instances, biological samples can be stained using a widevariety of stains and staining techniques. In some instances, thebiological sample is a tissue or a cell culture population that isincubated with a detectable marker (e.g., an antibody conjugated to afluorophore). In some instances, the biological sample is a section on aslide (e.g., a 10 μm section). In some instances, the biological sampleis dried after placement onto a glass slide. In some instances, thebiological sample is dried at 42° C. In some instances, drying occursfor about 1 hour, about 2, hours, about 3 hours, or until the sectionsbecome transparent. In some instances, the biological sample can bedried overnight (e.g., in a desiccator at room temperature).

In some embodiments, a sample can be stained using any number ofbiological stains, including but not limited to, acridine orange,Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin,ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine,methyl green, methylene blue, neutral red, Nile blue, Nile red, osmiumtetroxide, propidium iodide, rhodamine, or safranin. In some instances,the methods disclosed herein include imaging the biological sample. Insome instances, imaging the sample occurs prior to deaminating thebiological sample. In some instances, the sample can be stained usingknown staining techniques, including Can-Grunwald, Giemsa, hematoxylinand eosin (H&E), Jenner's, Leishman, Masson's trichrome, Papanicolaou,Romanowsky, silver, Sudan, Wright's, and/or Periodic Acid Schiff (PAS)staining techniques. PAS staining is typically performed after formalinor acetone fixation. In some instances, the stain is an H&E stain.

In some embodiments, the biological sample can be stained using adetectable label (e.g., radioisotopes, fluorophores, chemiluminescentcompounds, bioluminescent compounds, and dyes) as described elsewhereherein. In some embodiments, a biological sample is stained using onlyone type of stain or one technique. In some embodiments, stainingincludes biological staining techniques such as H&E staining. In someembodiments, staining includes identifying analytes usingfluorescently-conjugated antibodies. In some embodiments, a biologicalsample is stained using two or more different types of stains, or two ormore different staining techniques. For example, a biological sample canbe prepared by staining and imaging using one technique (e.g., H&Estaining and brightfield imaging), followed by staining and imagingusing another technique (e.g., IHC/IF staining and fluorescencemicroscopy) for the same biological sample.

In some embodiments, biological samples can be destained. Methods ofdestaining or discoloring a biological sample are known in the art, andgenerally depend on the nature of the stain(s) applied to the sample.For example, H&E staining can be destained by washing the sample in HCl,or any other acid (e.g., selenic acid, sulfuric acid, hydroiodic acid,benzoic acid, carbonic acid, malic acid, phosphoric acid, oxalic acid,succinic acid, salicylic acid, tartaric acid, sulfurous acid,trichloroacetic acid, hydrobromic acid, hydrochloric acid, nitric acid,orthophosphoric acid, arsenic acid, selenous acid, chromic acid, citricacid, hydrofluoric acid, nitrous acid, isocyanic acid, formic acid,hydrogen selenide, molybdic acid, lactic acid, acetic acid, carbonicacid, hydrogen sulfide, or combinations thereof). In some embodiments,destaining can include 1, 2, 3, 4, 5, or more washes in an acid (e.g.,HCl). In some embodiments, destaining can include adding HCl to adownstream solution (e.g., permeabilization solution). In someembodiments, destaining can include dissolving an enzyme used in thedisclosed methods (e.g., pepsin) in an acid (e.g., HCl) solution. Insome embodiments, after destaining hematoxylin with an acid, otherreagents can be added to the destaining solution to raise the pH for usein other applications. For example, SDS can be added to an aciddestaining solution in order to raise the pH as compared to the aciddestaining solution alone. As another example, in some embodiments, oneor more immunofluorescence stains are applied to the sample via antibodycoupling. Such stains can be removed using techniques such as cleavageof disulfide linkages via treatment with a reducing agent and detergentwashing, chaotropic salt treatment, treatment with antigen retrievalsolution, and treatment with an acidic glycine buffer. Methods formultiplexed staining and destaining are described, for example, inBolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin etal., Nat Commun. 2015; 6:8390, Pirici et al., J. Histochem. Cytochem.2009; 57:567-75, and Glass et al., J. Histochem. Cytochem. 2009;57:899-905, the entire contents of each of which are incorporated hereinby reference.

In some embodiments, immunofluorescence or immunohistochemistryprotocols (direct and indirect staining techniques) can be performed asa part of, or in addition to, the exemplary spatial workflows presentedherein. For example, tissue sections can be fixed according to methodsdescribed herein. The biological sample can be transferred to an array(e.g., capture probe array), wherein analytes (e.g., proteins) areprobed using immunofluorescence protocols. For example, the sample canbe rehydrated, blocked, and permeabilized (3×SSC, 2% BSA, 0.1% Triton X,1 U/μl RNAse inhibitor for 10 minutes at 4° C.) before being stainedwith fluorescent primary antibodies (1:100 in 3×SSC, 2% BSA, 0.1% TritonX, 1 U/μl RNAse inhibitor for 30 minutes at 4° C.). The biologicalsample can be washed, coverslipped (in glycerol+1 U/μl RNAse inhibitor),imaged (e.g., using a confocal microscope or other apparatus capable offluorescent detection), washed, and processed according to analytecapture or spatial workflows described herein.

In some instances, a glycerol solution and a cover slip can be added tothe sample. In some instances, the glycerol solution can include acounterstain (e.g., DAPI). As used herein, an antigen retrieval buffercan improve antibody capture in IF/IHC protocols. An exemplary protocolfor antigen retrieval can be preheating the antigen retrieval buffer(e.g., to 95° C.), immersing the biological sample in the heated antigenretrieval buffer for a predetermined time, and then removing thebiological sample from the antigen retrieval buffer and washing thebiological sample.

In some embodiments, optimizing permeabilization can be useful foridentifying intracellular analytes. Permeabilization optimization caninclude selection of permeabilization agents, concentration ofpermeabilization agents, and permeabilization duration. Tissuepermeabilization is discussed elsewhere herein.

In some embodiments, blocking an array and/or a biological sample inpreparation of labeling the biological sample decreases nonspecificbinding of the antibodies to the array and/or biological sample(decreases background). Some embodiments provide for blockingbuffers/blocking solutions that can be applied before and/or duringapplication of the label, wherein the blocking buffer can include ablocking agent, and optionally a surfactant and/or a salt solution. Insome embodiments, a blocking agent can be bovine serum albumin (BSA),serum, gelatin (e.g., fish gelatin), milk (e.g., non-fat dry milk),casein, polyethylene glycol (PEG), polyvinyl alcohol (PVA), orpolyvinylpyrrolidone (PVP), biotin blocking reagent, a peroxidaseblocking reagent, levamisole, Carnoy's solution, glycine, lysine, sodiumborohydride, pontamine sky blue, Sudan Black, trypan blue, FITC blockingagent, and/or acetic acid. The blocking buffer/blocking solution can beapplied to the array and/or biological sample prior to and/or duringlabeling (e.g., application of fluorophore-conjugated antibodies) to thebiological sample.

(b) Methods of Printing

Provided herein are methods for determining a location of an analyte ina cell, the method includes separating the cell from a plurality ofcells; printing the cell onto a surface comprising an array, wherein thearray comprises a plurality of capture probes, where a capture probe ofthe plurality of capture probes comprises (i) a spatial barcode and (ii)a capture domain; hybridizing the analyte to the capture domain; anddetermining (i) all or a part of the sequence of the analyte bound tothe capture domain, or a complement thereof, and (ii) all or a part ofthe sequence of the spatial barcode, or a complement thereof, and usingthe determined sequence of (i) and (ii) to identify the location of theanalyte in the cell. FIG. 7 shows an exemplary workflow for determininga location of an analyte in a cell.

The present disclosure provides methods for separating a cell from aplurality of cells and printing the cell onto a surface, where in someinstances the cell occupies a unique spatial location on the surfacethat is not occupied by any other cell in the plurality of cells. Inother word, the cell's location is unique to one or more spots on anarray. This provides the ability to examine both phenotype (e.g., usingimaging discussed herein) and genotype (e.g., by decoding the analytethat hybridizes to a capture probe).

In some embodiments, single-cell isolation techniques include, but arenot limited to, flow cytometry, laser microdissection, manual cellpicking, and microfluidics techniques. In some instances, the cells inthe sample may be aggregated, and may be disaggregated into individualcells using, for example, enzymatic or mechanical techniques. Examplesof enzymes used in enzymatic disaggregation include, but are not limitedto, dispase, collagenase, trypsin, or combinations thereof. Mechanicaldisaggregation can be performed, for example, using a tissuehomogenizer. In some instances of unaggregated cells or disaggregatedcells, the cells are distributed onto the substrate such that at leastone cell occupies a distinct spatial feature on the substrate. The cellscan be immobilized on the substrate (e.g., to prevent lateral diffusionof the cells). In some embodiments, a cell immobilization agent can beused to immobilize a non-aggregated or disaggregated sample on aspatially-barcoded array prior to analyte capture. A “cellimmobilization agent” can refer to an antibody, attached to a substrate,which can bind to a cell surface marker. In some embodiments, thedistribution of the plurality of cells on the substrate follows Poissonstatistics.

In some embodiments, cells from a plurality of cells are immobilized ona substrate. In some embodiments, the cells are immobilized to preventlateral diffusion, for example, by adding a hydrogel and/or by theapplication of an electric field.

Cell printing techniques are described herein and known in the art. See,e.g., Gross et al., Int. J. Mol. Sci. 16:16897-16919, 2015; Zhang etal., PNAS, 2014 111 (8) 2948-2953; Marzo et al., Nature Communications,6, 8661 (2015); Laurell et al., Chem. Soc. Rev., 2007,36, 492-506; andDing et al., PNAS, 2012 109 (28) 11105-11109, each of which isincorporated by reference in its entirety. Additional exemplary methodsof cell isolation include inkjet cell printing, surface engineering,physical constraints, microfluidic methods, or a combination thereof.

Inkjet cell printing may include encapsulating single cells in apicolitre-sized droplets that are then deposited via inkjet-likeprinting at defined locations. Inkjet cell printing can be carried outaccording to methods described in e.g., Yusof et al. Lab Chip11(14):2447-2454, 2011; Calvert, Science 318(5848):208-209, 2007; andNakamura et al. Tissue Eng 11(11-12): 1658-1666, 2005, each of which isincorporated by reference in its entirety.

In some instances, the methods provide herein isolate single cellsencapsulated in droplets (e.g., in a picoliter-sized droplet) that arethen deposited by inkjet-like printing at defined locations fordownstream genomic analysis. In some instances, the methods include useof a dispenser chip to print cells contained in a free flying droplet, acomputer vision system to detect single-cells inside the dispenser chipprior to printing, and appropriate automation equipment to printsingle-cells onto defined locations on a substrate (e.g., an array).

Surface engineering techniques can involve the use of, e.g.,micromagnetic substrates. See, e.g., those described in Tseng et al. NatMethods, 9(11):1113-1119, 2012, which is incorporated by reference inits entirety, and patterned substrates with fibronectin featuresgenerated using a tilted elastomeric pyramidal pen array (see, e.g.,those described in Giam et al. PNAS, 109(12):4377-4382, 2012, which isincorporated by reference in its entirety). Additional surfaceengineering techniques contemplated herein include those described inVermesh et al. Angew Chem Int Ed Engl, 50(32): 7378-7380, 2011; Aziouneet al. Lab Chip, 9(11):1640-1642, 2009; Tan et al. Integr Biol (Camb)1(10):587-594, 2009; Falconnet et al. Biomaterials, 27(16):3044-3063,2006; Suh et al. Biomaterials 25(3):557-563, 2004; Lee et al. Science,295(5560):1702-1705, 2002; and Chen et al. Science, 276(5317):1425-1428,1997, each of which is incorporated by reference in its entirety.

Physical constraints, such as those used in connection with microfluidictechniques, are useful for trapping single cells (See, e.g., Lin et al.Lab Chip, 13(4):714-721, 2013; and Chung et al. Anal Chem,83(18):7044-7052, 2011, each of which is incorporated by reference inits entirety). Physical constraints such as microwell arrays (See, e.g.Wood et al. PNAS, 107(22):10008-10013, 2010, which is incorporated byreference in its entirety); or those created using parylene membranes orelastomeric membranes can also be used for micro-patterning cells (See,e.g., Wright et al. J Biomed Mater Res A 85(2):530-538, 2008; Rosenthalet al. Biomaterials, 28(21):3208-3216, 2007; Rettig and Folch, AnalChem, 77(17):5628-5634, 2005; Ostuni et al. Langmuir 16(20):7811-7819,2000; and Folch et al. J Biomed Mater Res, 52(2):346-353, 2000, each ofwhich is incorporated by reference in its entirety).

Microfluidic techniques are useful either alone or in combination withany of the above techniques for separating a cell from a plurality ofcells and printing the cell on a surface. As an example, hydrodynamicmethods can passively steer individual cells in a continuous flow tomicro-patterned mechanical structures that spatially exclude more than adefined number of cells. Alternatively, active microfluidic techniquessuch as those employing optical, magnetic, electrical, and acousticforces can be used. In some embodiments, a plurality of cells can bespatially isolated and patterned in an acoustic field defined in twodimensions. The wavelength of the acoustic field can be of the sameorder as the cell dimensions, such that only one cell can inhabit agiven nodal location due to steric constraints. The wavelength can befrom about 10 μm to about 40 μm (e.g., about 12, 14, 16, 18, 20, 22, 24,26, 30, 35, or 40 μm). In some instances, surface acoustic waves (SAWs)at high frequency are used to create such a two-dimensional (2D)acoustic force field to spatially isolate a plurality of cells, whereone cell per acoustic well can be achieved (See, e.g., Collins et al.Nature Communications, 6:8686, 2015, which is incorporated by referencein its entirety). Cells trapped in acoustic wells can be held in placefor a certain period of time through the imposition of an externallyapplied acoustic field.

The methods of separating a cell from a plurality of cells providedherein can include filtering the cell through a mold. In some instances,the mold includes one or more chambers where optical, magnetic,electrical, or acoustic forces can be applied to spatially isolate thecell from a plurality of cells. The chambers can be made of any suitablematerials described herein and known in the art, and can be about 100 μmto about 1000 μm (e.g., about 200, 250, 280, 350, 400, 440, 480, 520,560, 600, 650, 700, 750, 800, 850, 900, or 950 μm) in length, and about100 μm to about 1000 μm (e.g., about 200, 250, 280, 350, 400, 440, 480,520, 560, 600, 650, 700, 750, 800, 850, 900, or 950 μm) in width.

In some instances, the mold includes a plurality of channels (e.g.,microfluidic channels) that steer the plurality of cells to flowthrough. At least 2, 5, 8, 15, 30, 100, 200, or 500 channels can beincluded in the mold, and the channels can be substantially parallel toeach other. In some embodiments, the mold includes from about 2 to about100 (e.g. about 5 to about 80, about 10 to about 60, or about 20 toabout 40) individual channels. The channels can have a width of about 25μm to about 50 μm (e.g., about 28, 30, 32, 34, 36, 38, 40, 42, 44, or 46μm), and a height of about 10 μm to about 15 μm (e.g., about 11, 12, 13,or 14 μm). A channel can include a plurality of traps along one side inthe interior of the channel, with an average distance between twoadjacent traps in the same channel being about 20 μm to about 200 μm(e.g., 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, or 180 μm), and theaverage distance between two closest traps in adjacent channels beingabout 20 μm to about 200 μm (e.g., 30, 40, 50, 60, 70, 80, 90, 100, 120,150, or 180 μm).

The traps can have any suitable size and shape (e.g., recesses orprotruding structures on one or both sides of a channel) that is capableof physically retaining a cell as it passes through the channel. In someinstances, the traps can have the shape of a hook. A trap can divide thewidth of the channel into a narrow side and a wide side, such that acell that passes through the narrow side is retained by the trap. Thewide side can have a gap that is larger than the narrow side by at least5 μm (e.g., at least 8, 10, or 12 μm). In some instances, once a cell isrestrained by a trap, the cell is retained for a sufficient amount oftime during which no other cell can be retained by the same trap.

In some instances, a plurality of cells suspended in culture medium areallowed to pass through the plurality of channels, and a subset of thecells are retained in the one or more traps within an individualchannel. The density of the cell suspension flown through an individualchannel can be at least 10⁶ cells per mL (e.g., at least 0.5×10⁷, 1×10⁷,1.5×10⁷, or 2×10⁷ cells per mL). In some instances, at least 50% (e.g.,at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%)of the traps are occupied by cells.

An individual channel can also include a first set of traps forcapturing cells that flow through the channel in a first direction, anda second set of traps for capturing cells that flow through the channelin the opposite direction.

Printing a cell separated from a plurality of cells onto a surface caninclude transferring the cell to any suitable surface described hereinor known in the art, and allowing the cell to adhere to the surface. Insome embodiments, the surface comprises a polystyrene, a glass, amodified glass, a functionalized glass, a hydrogel, a film, a membrane,a plastic, a nylon, a ceramic, a resin, Zeonor, silica, carbon, metals,inorganic glasses, optical fiber bundles, polymers, or a combinationthereof. In some instances, printing cells on a surface include allowingthe cells to adhere to the surface by incubation. The length ofincubation can be dependent on the adhesive capability of the cells tothe surface. In some embodiments, the cells are incubated for less than2 hours (e.g., less than 1.5, 1, or 0.5 hours). In some embodiments,separating a cell from the plurality of cells and printing the cell ontoa surface is completed under 2 hours (e.g., less than 1.5, 1, or 0.5hours).

In some instances, the methods provided herein include spatiallyisolating a plurality of cells and printing the cells onto a surface,such that two or more cells occupy unique locations on the surface assingle cells. The average distance between two adjacent single cellsfrom center to center can be between about 4 μm to about 150 μm (e.g.,about 5 to about 120, about 5 to about 100, about 5 to about 90, about 5to about 80, about 5 to about 60, about 5 to about 40, about 5 to about30, about 5 to about 20, about 20 to about 120, about 20 to about 100,about 20 to about 90, about 20 to about 80, about 20 to about 60, about20 to about 40, about 20 to about 30, about 30 to about 120, about 30 toabout 100, about 30 to about 90, about 30 to about 80, about 30 to about60, about 30 to about 40, about 40 to about 120, about 40 to about 100,about 40 to about 90, about 40 to about 80, about 40 to about 60, about60 to about 120, about 60 to about 100, about 60 to about 90, about 60to about 80, about 80 to about 120, about 80 to about 100, about 80 toabout 90, about 90 to about 120, about 90 to about 100, or about 100 to120 μm). In some instances, average distance between two adjacent singlecells from center to center is between about 5 to about 14 μm. The cellscan have a uniform or non-uniform distribution on the surface. In someinstances, the average distance between two adjacent cells in a firstportion of the surface is different from the average distance betweentwo adjacent cells in a second portion of the surface that does notoverlap with the first portion.

In some instances, the surface is part of the mold. For example, themold can include a chamber as described above which is formed in part bythe surface, e.g. by coupling a microfluidic chamber with the surface.As another example, the mold can include a plurality of channels asdescribed above formed by coupling a microfluidic device containing aplurality of channels with a suitable surface.

In some instances, the molds are removed following printing the cellonto the surface, thereby leaving the cell that has been separated fromthe plurality of cells adhered to the surface.

In some embodiments, the methods include preparing a suspension of cellsin culture medium, placing the cells in an inlet for a plurality ofmicrofluidic channels, and allowing the cells to flow into the channels.The cells can be pumped into the channels, and flow can be maintained bye.g. connecting an outlet for the microfluidic channels to anegative-pressure control system. A flow rate of less than 150 μm/s(e.g., less than 140, 130, 120, 110, or 100 μm/s) can be used. In someinstances, the flow rate is adjusted according to the rigidity of thecells. In some instances, at least 50% (e.g., at least 60%, 65%, 70%,75%, 80%, 85%, 90%, or 95%) of the traps have cells retained in them,before unanchored cells are washed away by replacing the cell suspensionwith culture medium. The negative pressure can be turned off to allowtrapped cells to adhere to the surface (e.g., by incubation). Celladhesion may occur after about 20 to 75 minutes of incubation. The moldcan be detached from the surface leaving the adhered cells on thesurface.

The methods provided herein can further include imaging the cell, e.g.,prior to separating the cell and printing the cell onto a surface, afterseparating the cell but before printing the cell onto a surface, orafter cell is printed onto a surface. Imaging a cell can be performedusing any technique as described herein.

(c) Detecting an Analyte in a Cell

The present disclosure provides methods for detecting an analyte from acell printed onto a surface, where the cell has been separated from aplurality of cells (e.g., by filtering through a mold); the surfacecomprising an array comprising a plurality of capture probes, where acapture probe of the plurality of capture probes comprises: (i) aspatial barcode (e.g., any of the spatial barcodes described herein) and(ii) a capture domain (e.g. any of the capture domains describedherein). The methods further include hybridizing the analyte to thecapture domain; and determining (i) all or a part of the sequence of theanalyte bound to the capture domain, or a complement thereof, and (ii)all or a part of the sequence of the spatial barcode, or a complementthereof, and using the determined sequence of (i) and (ii) to identifythe location of the analyte in the cell.

The apparatus, systems, methods, and compositions described in thisdisclosure can be used to detect and analyze a wide variety of differentanalytes. For the purpose of this disclosure, an “analyte” can includeany biological substance, structure, moiety, or component to beanalyzed. The term “target” can similarly refer to an analyte ofinterest. Analytes can be broadly classified into one of two groups:nucleic acid analytes, and non-nucleic acid analytes. Examples ofnon-nucleic acid analytes include, but are not limited to, lipids,carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked),lipoproteins, phosphoproteins, specific phosphorylated or acetylatedvariants of proteins, amidation variants of proteins, hydroxylationvariants of proteins, methylation variants of proteins, ubiquitylationvariants of proteins, sulfation variants of proteins, viral coatproteins, extracellular and intracellular proteins, antibodies, andantigen binding fragments. In some embodiments, the analyte can be anorganelle (e.g., nuclei or mitochondria).

The biological analyte can be any suitable biological analyte describedherein and known in the art, including but not limited to: protein, ananalyte comprising a post-translational modification, and nucleic acid(e.g., DNA or RNA). In some instances, the biological analyte is anmRNA. Releasing the biological analyte so that it can hybridize to thecapture domain can include permeabilizing the cell using techniquesdescribed herein and known in the art. Exemplary methods include:electrophoresis and administration of a permeabilization agent. Thecells can optionally be fixed or stained using methods described hereinprior to permeabilization. In some instances, the cells are stainedafter fixing. The methods can further include imaging the cell, e.g.,prior to or after releasing hybridizing the biological analyte from thecell to the capture domain. In some instances, imaging is helpful indetermining the morphology of the cell, or determining the label ormarker on the cell.

Analytes can be derived from a specific type of cell and/or a specificsub-cellular region. For example, analytes can be derived from cytosol,from cell nuclei, from mitochondria, from microsomes, and moregenerally, from any other compartment, organelle, or portion of a cell.Permeabilizing agents that specifically target certain cell compartmentsand organelles can be used to selectively release analytes from cellsfor analysis.

Examples of nucleic acid analytes include DNA analytes such as genomicDNA, methylated DNA, specific methylated DNA sequences, fragmented DNA,mitochondrial DNA, in situ synthesized PCR products, and RNA/DNAhybrids.

Examples of nucleic acid analytes also include RNA analytes such asvarious types of coding and non-coding RNA. Examples of the differenttypes of RNA analytes include messenger RNA (mRNA), ribosomal RNA(rRNA), transfer RNA (tRNA), microRNA (miRNA), and viral RNA. The RNAcan be a transcript (e.g., present in a tissue section). The RNA can besmall (e.g., less than 200 nucleic acid bases in length) or large (e.g.,RNA greater than 200 nucleic acid bases in length). Small RNAs mainlyinclude 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA),microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA(snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA),and small rDNA-derived RNA (srRNA). The RNA can be double-stranded RNAor single-stranded RNA. The RNA can be circular RNA. The RNA can be abacterial rRNA (e.g., 16s rRNA or 23s rRNA).

Capture probes can detect nucleic acid and non-nucleic acid (e.g.,protein) analytes. In the setting of detecting a nucleic acid, anexemplary probe in FIG. 1 and described above hybridize to anindiscriminant sequence (e.g., a poly(A) tail) of a nucleic acidanalyte. In the setting of detecting a non-nucleic acid (e.g., aprotein), an analyte binding moiety binds to a protein of interest.Then, the analyte binding moiety is affixed to a nucleic acid sequencethat can hybridize to the capture probe. As described above, FIG. 4provides an exemplary embodiment of hybridization of an analyte captureagent to a capture probe using a nucleic acid sequence.

After an analyte from the sample has hybridized or otherwise beenassociated with a capture probe according to any of the methodsdescribed above in connection with the general spatial cell-basedanalytical methodology, the barcoded constructs that result fromhybridization/association are analyzed.

The density of the capture probes on the substrate array can be uniformor non-uniform. In some embodiments, the density of capture probes on afirst portion of the substrate array is lower than the density ofcapture probes on a second portion of the array. In instances where aplurality of cells are separated and printed onto a surface to generatean array of cells, the methods provided herein include contacting thearray of cells with a substrate comprising an array having comprising aplurality of capture probes, such that at least one capture probe is incontact with a cell on the array.

In some embodiments, after contacting a biological sample with asubstrate that includes capture probes, a removal step can optionally beperformed to remove all or a portion of the biological sample from thesubstrate. In some embodiments, the removal step includes enzymaticand/or chemical degradation of cells of the biological sample. Forexample, the removal step can include treating the biological samplewith an enzyme (e.g., a proteinase, e.g., proteinase K) to remove atleast a portion of the biological sample from the substrate. In someembodiments, the removal step can include ablation of the tissue (e.g.,laser ablation).

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample), the method comprising: (a) optionally stainingand/or imaging a biological sample on a substrate; (b) permeabilizing(e.g., providing a solution comprising a permeabilization reagent to)the biological sample on the substrate; (c) contacting the biologicalsample with an array comprising a plurality of capture probes, wherein acapture probe of the plurality captures the biological analyte; and (d)analyzing the captured biological analyte, thereby spatially detectingthe biological analyte; wherein the biological sample is fully orpartially removed from the substrate.

In some embodiments, a biological sample is not removed from thesubstrate. For example, the biological sample is not removed from thesubstrate prior to releasing a capture probe (e.g., a capture probebound to an analyte) from the substrate. In some embodiments, suchreleasing comprises cleavage of the capture probe from the substrate(e.g., via a cleavage domain). In some embodiments, such releasing doesnot comprise releasing the capture probe from the substrate (e.g., acopy of the capture probe bound to an analyte can be made and the copycan be released from the substrate, e.g., via denaturation). In someembodiments, the biological sample is not removed from the substrateprior to analysis of an analyte bound to a capture probe after it isreleased from the substrate. In some embodiments, the biological sampleremains on the substrate during removal of a capture probe from thesubstrate and/or analysis of an analyte bound to the capture probe afterit is released from the substrate. In some embodiments, the biologicalsample remains on the substrate during removal (e.g., via denaturation)of a copy of the capture probe (e.g., complement). In some embodiments,analysis of an analyte bound to capture probe from the substrate can beperformed without subjecting the biological sample to enzymatic and/orchemical degradation of the cells (e.g., permeabilized cells) orablation of the tissue (e.g., laser ablation).

In some embodiments, at least a portion of the biological sample is notremoved from the substrate. For example, a portion of the biologicalsample can remain on the substrate prior to releasing a capture probe(e.g., a capture probe bound to an analyte) from the substrate and/oranalyzing an analyte bound to a capture probe released from thesubstrate. In some embodiments, at least a portion of the biologicalsample is not subjected to enzymatic and/or chemical degradation of thecells (e.g., permeabilized cells) or ablation of the tissue (e.g., laserablation) prior to analysis of an analyte bound to a capture probe fromthe substrate.

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample) that include: (a) optionally staining and/or imaginga biological sample on a substrate; (b) permeabilizing (e.g., providinga solution comprising a permeabilization reagent to) the biologicalsample on the substrate; (c) contacting the biological sample with anarray comprising a plurality of capture probes, wherein a capture probeof the plurality captures the biological analyte; and (d) analyzing thecaptured biological analyte, thereby spatially detecting the biologicalanalyte; where the biological sample is not removed from the substrate.

In some embodiments, provided herein are methods for spatially detectinga biological analyte of interest from a biological sample that include:(a) staining and imaging a biological sample on a substrate; (b)providing a solution comprising a permeabilization reagent to thebiological sample on the substrate; (c) contacting the biological samplewith an array on a substrate, wherein the array comprises one or morecapture probe pluralities thereby allowing the one or more pluralitiesof capture probes to capture the biological analyte of interest; and (d)analyzing the captured biological analyte, thereby spatially detectingthe biological analyte of interest; where the biological sample is notremoved from the substrate.

In some embodiments, the method further includes subjecting a region ofinterest in the biological sample to spatial transcriptomic analysis. Insome embodiments, one or more of the capture probes includes a capturedomain. In some embodiments, one or more of the capture probes comprisesa unique molecular identifier (UMI). In some embodiments, one or more ofthe capture probes comprises a cleavage domain. In some embodiments, thecleavage domain comprises a sequence recognized and cleaved by auracil-DNA glycosylase, apurinic/apyrimidinic (AP) endonuclease (APE1),U uracil-specific excision reagent (USER), and/or an endonuclease VIII.In some embodiments, one or more capture probes do not comprise acleavage domain and is not cleaved from the array.

In some embodiments, a capture probe can be extended (an “extendedcapture probe,” e.g., as described herein). For example, extending acapture probe can include generating cDNA from a captured (hybridized)RNA. This process involves synthesis of a complementary strand of thehybridized nucleic acid, e.g., generating cDNA based on the captured RNAtemplate (the RNA hybridized to the capture domain of the captureprobe). Thus, in an initial step of extending a capture probe, e.g., thecDNA generation, the captured (hybridized) nucleic acid, e.g., RNA, actsas a template for the extension, e.g., reverse transcription, step.

In some embodiments, the capture probe is extended using reversetranscription. For example, reverse transcription includes synthesizingcDNA (complementary or copy DNA) from RNA, e.g., (messenger RNA), usinga reverse transcriptase. In some embodiments, reverse transcription isperformed while the tissue is still in place, generating an analytelibrary, where the analyte library includes the spatial barcodes fromthe adjacent capture probes. In some embodiments, the capture probe isextended using one or more DNA polymerases.

In some embodiments, a capture domain of a capture probe includes aprimer for producing the complementary strand of a nucleic acidhybridized to the capture probe, e.g., a primer for DNA polymeraseand/or reverse transcription. The nucleic acid, e.g., DNA and/or cDNA,molecules generated by the extension reaction incorporate the sequenceof the capture probe. The extension of the capture probe, e.g., a DNApolymerase and/or reverse transcription reaction, can be performed usinga variety of suitable enzymes and protocols.

In some embodiments, a full-length DNA (e.g., cDNA) molecule isgenerated. In some embodiments, a “full-length” DNA molecule refers tothe whole of the captured nucleic acid molecule. However, if a nucleicacid (e.g., RNA) was partially degraded in the tissue sample, then thecaptured nucleic acid molecules will not be the same length as theinitial RNA in the tissue sample. In some embodiments, the 3′ end of theextended probes, e.g., first strand cDNA molecules, is modified. Forexample, a linker or adaptor can be ligated to the 3′ end of theextended probes. This can be achieved using single stranded ligationenzymes such as T4 RNA ligase or Circligase™ (available from Lucigen,Middleton, Wis.). In some embodiments, template switchingoligonucleotides are used to extend cDNA in order to generate afull-length cDNA (or as close to a full-length cDNA as possible). Insome embodiments, a second strand synthesis helper probe (a partiallydouble stranded DNA molecule capable of hybridizing to the 3′ end of theextended capture probe), can be ligated to the 3′ end of the extendedprobe, e.g., first strand cDNA, molecule using a double strandedligation enzyme such as T4 DNA ligase. Other enzymes appropriate for theligation step are known in the art and include, e.g., Tth DNA ligase,Taq DNA ligase, Thermococcus sp. (strain 9° N) DNA ligase (9° N™ DNAligase, New England Biolabs), Ampligase™ (available from Lucigen,Middleton, Wis.), and SplintR (available from New England Biolabs,Ipswich, Mass.). In some embodiments, a polynucleotide tail, e.g., apoly(A) tail, is incorporated at the 3′ end of the extended probemolecules. In some embodiments, the polynucleotide tail is incorporatedusing a terminal transferase active enzyme.

In some embodiments, double-stranded extended capture probes are treatedto remove any unextended capture probes prior to amplification and/oranalysis, e.g., sequence analysis. This can be achieved by a variety ofmethods, e.g., using an enzyme to degrade the unextended probes, such asan exonuclease enzyme, or purification columns.

In some embodiments, extended capture probes are amplified to yieldquantities that are sufficient for analysis, e.g., via DNA sequencing.In some embodiments, the first strand of the extended capture probes(e.g., DNA and/or cDNA molecules) acts as a template for theamplification reaction (e.g., a polymerase chain reaction).

In some embodiments, the amplification reaction incorporates an affinitygroup onto the extended capture probe (e.g., RNA-cDNA hybrid) using aprimer including the affinity group. In some embodiments, the primerincludes an affinity group and the extended capture probes includes theaffinity group. The affinity group can correspond to any of the affinitygroups described previously.

In some embodiments, the extended capture probes including the affinitygroup can be coupled to a substrate specific for the affinity group. Insome embodiments, the substrate can include an antibody or antibodyfragment. In some embodiments, the substrate includes avidin orstreptavidin and the affinity group includes biotin. In someembodiments, the substrate includes maltose and the affinity groupincludes maltose-binding protein. In some embodiments, the substrateincludes maltose-binding protein and the affinity group includesmaltose. In some embodiments, amplifying the extended capture probes canfunction to release the extended probes from the surface of thesubstrate, insofar as copies of the extended probes are not immobilizedon the substrate.

In some embodiments, the extended capture probe or complement oramplicon thereof is released. The step of releasing the extended captureprobe or complement or amplicon thereof from the surface of thesubstrate can be achieved in a number of ways. In some embodiments, anextended capture probe or a complement thereof is released from thearray by nucleic acid cleavage and/or by denaturation (e.g., by heatingto denature a double-stranded molecule).

In some embodiments, the extended capture probe or complement oramplicon thereof is released from the surface of the substrate (e.g.,array) by physical means. For example, where the extended capture probeis indirectly immobilized on the array substrate, e.g., viahybridization to a surface probe, it can be sufficient to disrupt theinteraction between the extended capture probe and the surface probe.Methods for disrupting the interaction between nucleic acid moleculesinclude denaturing double stranded nucleic acid molecules are known inthe art. A straightforward method for releasing the DNA molecules (i.e.,of stripping the array of extended probes) is to use a solution thatinterferes with the hydrogen bonds of the double stranded molecules. Insome embodiments, the extended capture probe is released by an applyingheated solution, such as water or buffer, of at least 85° C., e.g., atleast 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99° C. In some embodiments,a solution including salts, surfactants, etc. that can furtherdestabilize the interaction between the nucleic acid molecules is addedto release the extended capture probe from the substrate.

In some embodiments, where the extended capture probe includes acleavage domain, the extended capture probe is released from the surfaceof the substrate by cleavage. For example, the cleavage domain of theextended capture probe can be cleaved by any of the methods describedherein. In some embodiments, the extended capture probe is released fromthe surface of the substrate, e.g., via cleavage of a cleavage domain inthe extended capture probe, prior to the step of amplifying the extendedcapture probe.

In some embodiments, probes complementary to the extended capture probecan be contacted with the substrate. In some embodiments, the biologicalsample can be in contact with the substrate when the probes arecontacted with the substrate. In some embodiments, the biological samplecan be removed from the substrate prior to contacting the substrate withprobes. In some embodiments, the probes can be labeled with a detectablelabel (e.g., any of the detectable labels described herein). In someembodiments, probes that do not specially bind (e.g., hybridize) to anextended capture probe can be washed away. In some embodiments, probescomplementary to the extended capture probe can be detected on thesubstrate (e.g., imaging, any of the detection methods describedherein).

In some embodiments, probes complementary to an extended capture probecan be about 4 nucleotides to about 100 nucleotides long. In someembodiments, probes (e.g., detectable probes) complementary to anextended capture probe can be about 10 nucleotides to about 90nucleotides long. In some embodiments, probes (e.g., detectable probes)complementary to an extended capture probe can be about 20 nucleotidesto about 80 nucleotides long. In some embodiments, probes (e.g.,detectable probes) complementary to an extended capture probe can beabout 30 nucleotides to about 60 nucleotides long. In some embodiments,probes (e.g., detectable probes) complementary to an extended captureprobe can be about 40 nucleotides to about 50 nucleotides long. In someembodiments, probes (e.g., detectable probes) complementary to anextended capture probe can be about 5, about 6, about 7, about 8, about9, about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, about 25, about 26, about 27, about 28, about 29, about 30,about 31, about 32, about 33, about 34, about 35, about 36, about 37,about 38, about 39, about 40, about 41, about 42, about 43, about 44,about 45, about 46, about 47, about 48, about 49, about 50, about 51,about 52, about 53, about 54, about 55, about 56, about 57, about 58,about 59, about 60, about 61, about 62, about 63, about 64, about 65,about 66, about 67, about 68, about 69, about 70, about 71, about 72,about 73, about 74, about 75, about 76, about 77, about 78, about 79,about 80, about 81, about 82, about 83, about 84, about 85, about 86,about 87, about 88, about 89, about 90, about 91, about 92, about 93,about 94, about 95, about 96, about 97, about 98, and about 99nucleotides long.

In some embodiments, about 1 to about 100 probes can be contacted to thesubstrate and specifically bind (e.g., hybridize) to an extended captureprobe. In some embodiments, about 1 to about 10 probes can be contactedto the substrate and specifically bind (e.g., hybridize) to an extendedcapture probe. In some embodiments, about 10 to about 100 probes can becontacted to the substrate and specifically bind (e.g., hybridize) to anextended capture probe. In some embodiments, about 20 to about 90 probescan be contacted to the substrate and specifically bind (e.g.,hybridize) to an extended capture probe. In some embodiments, about 30to about 80 probes (e.g., detectable probes) can be contacted to thesubstrate and specifically bind (e.g., hybridize) to an extended captureprobe. In some embodiments, about 40 to about 70 probes can be contactedto the substrate and specifically bind (e.g., hybridize) to an extendedcapture probe. In some embodiments, about 50 to about 60 probes can becontacted to the substrate and specifically bind (e.g., hybridize) to anextended capture probe. In some embodiments, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 11, about12, about 13, about 14, about 15, about 16, about 17, about 18, about19, about 20, about 21, about 22, about 23, about 24, about 25, about26, about 27, about 28, about 29, about 30, about 31, about 32, about33, about 34, about 35, about 36, about 37, about 38, about 39, about40, about 41, about 42, about 43, about 44, about 45, about 46, about47, about 48, about 49, about 50, about 51, about 52, about 53, about54, about 55, about 56, about 57, about 58, about 59, about 60, about61, about 62, about 63, about 64, about 65, about 66, about 67, about68, about 69, about 70, about 71, about 72, about 73, about 74, about75, about 76, about 77, about 78, about 79, about 80, about 81, about82, about 83, about 84, about 85, about 86, about 87, about 88, about89, about 90, about 91, about 92, about 93, about 94, about 95, about96, about 97, about 98, and about 99 probes can be contacted to thesubstrate and specifically bind (e.g., hybridize) to an extended captureprobe.

In some embodiments, the probes can be complementary to a single analyte(e.g., a single gene). In some embodiments, the probes can becomplementary to one or more analytes (e.g., analytes in a family ofgenes). In some embodiments, the probes (e.g., detectable probes) can befor a panel of genes associated with a disease (e.g., cancer,Alzheimer's disease, Parkinson's disease).

In some instances, the analyte and capture probe can be amplified orcopied, creating a plurality of cDNA molecules. In some embodiments,cDNA can be denatured from the capture probe template and transferred(e.g., to a clean tube) for amplification, and/or library construction.The spatially-barcoded cDNA can be amplified via PCR prior to libraryconstruction. The cDNA can then be enzymatically fragmented andsize-selected in order to optimize for cDNA amplicon size. P5 and P7sequences directed to capturing the amplicons on a sequencing flowcell(Illumina sequencing instruments) can be appended to the amplicons, i7,and i5 can be used as sample indexes, and TruSeq Read 2 can be added viaEnd Repair, A-tailing, Adaptor Ligation, and PCR. The cDNA fragments canthen be sequenced using paired-end sequencing using TruSeq Read 1 andTruSeq Read 2 as sequencing primer sites. The additional sequences aredirected toward Illumina sequencing instruments or sequencinginstruments that utilize those sequences; however a skilled artisan willunderstand that additional or alternative sequences used by othersequencing instruments or technologies are also equally applicable foruse in the aforementioned methods.

In some embodiments, where a sample is barcoded directly viahybridization with capture probes or analyte capture agents hybridized,bound, or associated with either the cell surface, or introduced intothe cell, as described above, sequencing can be performed on the intactsample.

A wide variety of different sequencing methods can be used to analyzebarcoded analytes (e.g., the ligation product). In general, sequencedpolynucleotides can be, for example, nucleic acid molecules such asdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA), includingvariants or derivatives thereof (e.g., single stranded DNA or DNA/RNAhybrids, and nucleic acid molecules with a nucleotide analog).

Sequencing of polynucleotides can be performed by various systems. Moregenerally, sequencing can be performed using nucleic acid amplification,polymerase chain reaction (PCR) (e.g., digital PCR and droplet digitalPCR (ddPCR), quantitative PCR, real time PCR, multiplex PCR, PCR-basedsingle plex methods, emulsion PCR), and/or isothermal amplification.Non-limiting examples of methods for sequencing genetic materialinclude, but are not limited to, DNA hybridization methods (e.g.,Southern blotting), restriction enzyme digestion methods, Sangersequencing methods, next-generation sequencing methods (e.g.,single-molecule real-time sequencing, nanopore sequencing, and Polonysequencing), ligation methods, and microarray methods.

EXAMPLES Example 1. Printing of Cells and Spatial Analysis of Analytes

A population of cells is cultured in an in vitro system (e.g., in a6-well plate) in growth media known in the art (e.g., media comprising1×DMEM and 10% fetal bovine serum). The cells are washed using 1×PBS andare trypsinized, allowing for separation of cells. Viability of cells ismeasured using methods known in the art (e.g., using trypan blue). Cellsare filtered through a mold to allow for individual placement onto anarray. The array comprises a plurality of capture probes.

After adhering the cells onto the array, the cells are imaged. In someinstances, the cells are incubated with detectable markers in order tophenotypically analyze the cell. Cells are imaged using brightfield orfluorescent microscopy.

Cells printed onto an array are permeabilized using a solutioncomprising proteinase K, and analytes are captured by capture probes onthe array. Analytes that hybridize to the capture probes are thenextended. The extended capture probes are denatured. Denatured, extendedcapture probes are indexed and the amplified libraries are subjected toquality control before being sequenced.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for determining a location of an analytein a cell, the method comprising: (a) separating the cell from aplurality of cells by filtering the plurality of cells through a networkof microfluidic channels, wherein the cell is filtered through amicrofluidic channel of the network that allows the cell to migratetowards a surface comprising an array; (b) dispensing the cell onto thesurface comprising the array, wherein the microfluidic channel of thenetwork dispenses the cell onto a unique area of the surface, whereinthe array comprises a plurality of capture probes, wherein a captureprobe of the plurality of capture probes comprises: (i) a spatialbarcode and (ii) a capture domain; (c) hybridizing the analyte to thecapture domain; and (d) determining (i) all or a part of the sequence ofthe analyte bound to the capture domain, or a complement thereof, and(ii) the spatial barcode, or a complement thereof, and using thedetermined sequences of (i) and (ii) to identify the location of theanalyte in the cell.
 2. The method of claim 1, wherein the step ofseparating the cell from a plurality of cells comprises filtering thecell through a mold.
 3. The method of claim 2, further comprisingremoving the mold after printing the cell onto the surface.
 4. Themethod of claim 1, wherein the plurality of cells has at least 80%viability.
 5. The method of claim 1, wherein the cell is from aheterogeneous cell population.
 6. The method of claim 1, wherein thecell is from a formalin-fixed, paraffin-embedded (FFPE) sample, a frozensample, or a fresh sample.
 7. The method of claim 1, wherein the cell isfrom a tissue sample or a cell culture sample.
 8. The method of claim 1,wherein the surface comprises glass, a modified glass, a functionalizedglass, a hydrogel, a film, a membrane, a plastic, a nylon, a ceramic, aresin, Zeonor, silica, carbon, metals, inorganic glasses, optical fiberbundles, polymers, or combinations thereof.
 9. The method of claim 2,wherein the mold comprises the network of microfluidic channels.
 10. Themethod of claim 9, wherein the mold comprises from about 2 to about 100individual microfluidic channels in the network of microfluidicchannels.
 11. The method of claim 10, wherein the individualmicrofluidic channels comprise trap spacing, wherein the trap spacingsin individual microfluidic channels proximal to the surface are narrowerin diameter than the trap spacings in individual microfluidic channelsdistal to the surface.
 12. The method of claim 1, wherein the analytecomprises DNA or RNA.
 13. The method of claim 12, wherein the RNA is anmRNA molecule.
 14. The method of claim 1, wherein the determining stepcomprises amplifying all or part of the analyte bound to the capturedomain.
 15. The method of claim 1, wherein the determining stepcomprises sequencing.
 16. The method of claim 1, further comprisingimaging the cell.
 17. The method of claim 16, wherein the imaging isused to determine morphology of the cell.
 18. The method of claim 1,wherein the capture probe comprises a unique molecular identifier, acleavage domain, and/or a functional domain.